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Background

Heavy metals used in industrial and household applications can pose harmful health
effects. Tungsten alloys (WAs) have been widely used in many engineering, automotive
and marine applications. In military operations, WAs containing tungsten (W; 91% w/w),
nickel (Ni; 6% w/w) and cobalt (Co; 3% w/w) have been deployed in armor-penetrating
munitions as substitutes for depleted uranium. Despite widespread use, a clear understanding
of the potential effects of WAs on physiological processes and gene expression is
not available. WAs have carcinogenic potential as demonstrated by cancer development
in rats with intramuscular implanted WA pellets. This suggests a potential involvement
of epigenetic events previously implicated as environmental triggers of cancer. In
the present study, we have attempted to unravel WA-mediated alterations in gene expression
and gene-specific epigenetic modifications at the genome-wide level.

Materials and methods

C2C12 (mouse myoblast) cell cultures were exposed to 1,000 µM WA for 24 h. ChIP assays
were performed with cross-linked cells from WA-treated and untreated C2C12 cultures
using anti-RNA Pol II, anti-phospho histone H3Ser10, anti-trimethyl histone H3K4 and
anti-trimethyl histone H3K27 antibodies. ChIP assays were followed by sequencing where
single-ended 50 bp reads were generated using a combination of the Applied Biosystems
SOLiD™ and Illumina Hi-Seq™ systems. Data analysis was done using in-house developed
pipelines (PERL/Unix) as well as several open source bioinformatics software from
R/Bioconductor project and other sources.

Results

Using different in vitro models, we examined metal-induced cytotoxicity and epigenetic modifications where
WA showed cytotoxicity at concentrations >50 µg/ml, with C2C12 being relatively resistant
to WA-mediated toxic impact. Using ChIP-Seq, we found several histone modifications
up- and downregulated in the promoter regions of genes related to learning and memory
mechanisms, with maximum impact observed for H3Ser10 phosphorylation. A total of 101
regions in the mouse genome were found to be most significantly depleted of H3S10
phosphorylation after WA treatment (fold change >3), these targets included several
genes with neurological functions including voltage-dependent calcium channel (CACNB1), phosphatidylinositol 4-kinase type 2 (Pi4k2a) and Kinesin 5A (KIF5A). In addition, pathway analysis of these 101 regions revealed 15 genes as part of regulatory
networks that are responsible for developmental disorders, hereditary disorders and
neurological diseases. ChIP-Seq analysis of other histone modifications and Pol II
binding patterns are still ongoing. We plan to correlate expression levels investigated
by RNA-Seq and epigenetic profiles impacted by WA exposure.

Conclusions

Our results reveal epigenetic modifications triggered by WA exposure in C2C12 cells
for the first time at a genome-wide level. In addition to epigenetic changes observed
for specific genes, ChIP-Seq analysis confirmed our previous report on the gross genomic
depletion of H3Ser10 phosphorylation [1]. Future investigations on genes identified in this study will help unravel the mechanisms
involved in WA toxicity that may lead towards the development of therapeutics.